Cultures of permanent lines of human promyelocytic cells and their uses for the screening of molecules utilizable in particular in the treatment of leukemias

ABSTRACT

The invention concerns permanent cell lines of human promyelocyte cells characteristic of acute promyelocytic leukemias, these cells being cytogenetically characterized by a translocation t(15;17), capable of indefinite proliferation in a culture medium. It also concerns the use of these cell lines, particularly for screening molecules capable of inducing cell maturation.

This application was filed under 35 U.S.C. 371 as the national stage ofPCT/FR92/00173 filed Feb. 25, 1992, and published as WO92/14815 Sep. 3,1992.

Acute promyelocytic leukemia (or APL or also leukemia type 3) is awell-defined variety of leukemia, and is cytogenetically characterizedby a translocation t (15; 17) (q22; q11-12) (Rowley J. D. et al., Lancet1:549, 1977). This translocation could play a role in the uncontrolledcellular proliferation with blockage of cell maturation which occurs inAPL.

In vitro and in vivo studies have made it possible to demonstrate thatretinoic acid (also designated hereafter as RA) stops the process ofcellular proliferation of the APL and induces the morphological andfunctional maturation of the promyelocytes towards the granulocytestage.

This effect of retinoic acid on the promyelocytes suggests that theexpression of certain genes intimately implicated in the transduction ofthe RA signal or in the process of maturation of the cells is adverselyaffected by the translocation mentioned above.

In vitro studies conducted on promyelocytes derived from patientssuffering from APL have made it possible to demonstrate that the genecoding for the alpha receptor of retinoic acid (RARα receptor) normallylocated on chromosome 17 has been rearranged by translocation with thePML locus normally located on chromosome 15 (de The et al., Nature, 347,pp558-561, 1990). By using probes corresponding to segments of DNAlocalized in the vicinity of the breaking points of the translocation,it was possible to define genomic rearrangements of one or other locusin the patients suffering from APL mentioned above. Consequently, theRARα and PML genes are rearranged in the APLs. These experimentsstrongly support the implication of the alpha receptor of retinoic acidin acute promyelocytic leukemias.

In the light of the importance of the role played by the RARα in thepathology of the APLs, it would be particularly interesting to be ableto have available cell lines constituted of promyelocytes of humanorigin which have this translocation t (15; 17) characteristic of theAPLs in order in particular to screen molecules derived from retinoicacid, or other molecules capable of restoring cell maturation withoutnonetheless exhibiting the toxic effects of retinoic acid.

However, it has hitherto not been possible to maintain a line of humanpromyelocytic cells in culture and, consequently, it has not beenpossible to perform this type of study.

Although the HL60 line has been called "promyelocytic" (Gallagher R. etal., Blood 54:713, 1979), it was subsequently recognized that, on theone hand, it is derived from a myeloblastic leukemia characterized bycells with a certain degree of maturation (M2) (Dalton W. T. et al.,Blood 71; 242, 1988) and, on the other, it does not contain theabove-mentioned translocation t (15; 17).

One of the objectives of the present invention is precisely that ofmaking available a cell culture which permits the screening of moleculescapable of restoring cellular maturation.

The subject of the present invention is permanent cell lines,characterized in that they are constituted essentially of promyelocyticcells characteristic of acute human promyelocytic leukemias, these cellsbeing cytogenetically characterized by a translocation t (15; 17), andcapable of proliferating indefinitely in a culture medium.

The culture medium in which the above-mentioned cells are capable ofproliferating is advantageously constituted by a synthetic nutrientmedium (RPMI 1640) and fetal calf serum.

The cell lines of the invention and their variants are characterizedparticularly in that they possess DNA fingerprints on gelelectrophoresis identical, wholly or in part, with the DNA fingerprintshown in FIG. 1.

As an illustration, the DNA fingerprints of the cells constituting thecell lines of the invention are obtained by the procedure defined inNucleic Acids Research (1988), vol. 16, No. 9, p. 4161.

The cell lines of the invention are advantageously characterized in thatthey are either sensitive to retinoic acid, i.e. capable ofdifferentiating from the promyelocytic stage towards the granulocytestage under the influence of retinoic acid, or, on the contrary, areresistant to retinoic acid and are thus incapable of differentiatingunder the influence of this latter or also are capable ofdifferentiating as a result of treatment by retinoic acid associatedwith one or more reducing agents of such differentiation.

The invention relates to a cell line sensitive to retinoic acid (i.e.the majority of the cells of which are capable of differentiating underthe influence of this latter), and which was deposited with theCollection Nationale de Culture de Micro-organismes de l'INSTITUTPASTEUR (CNCM) on 25 Feb. 1991 under the number I-1045, as well as withthe Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSM)on 14 Feb. 1992 under the number DSM.ACC.2030.

This cell line was obtained from a patient suffering from an APL, whohad been treated by chemotherapy and was in a relapse phase. Cells ofthe bone marrow were taken from this patient and placed in culture on alayer of cells of the bone marrow stroma (Lanotte et al., J. Cell. Sci.50:281, 1981) until an autonomous cell line was established (designatedhereafter by the expression NB4). This cell line NB4 is characterized inparticular by the DNA fingerprint shown in FIG. 1, and is constitutedmainly of cells sensitive to retinoic acid. It forms the subject of amore thorough study in the detailed description of the invention whichfollows.

The subject of the invention is also a cell line resistant to retinoicacid (i.e. the majoity of the cells of which are incapable ofdifferentiating under the influence of the latter), this line(designated hereafter by the expression NB4R) being obtained byincubation of the previously mentioned NB4 line in the presence ofall-trans retinoic acid, followed by the isolation of the resistantcells and the amplification of these latter still in the presence ofretinoic acid.

The invention also relates to a screening procedure for moleculescapable of restoring cellular maturation, and more particularly ofinducing the maturation of the promyelocytes towards the granulocytestage, in particular in the context of acute human promyelocyticleukemias, this procedure being characterized in that it comprises theplacing of a specific quantity of the molecule under study in contactwith a cell line of the invention, and more particularly with asensitive cell line, followed by the detection of possibledifferentiation of the cells of the said line.

The subject of the invention is also a screening procedure for moleculescapable of abolishing the resistance which the cells exhibit, and moreparticularly cells characteristic of the acute human promyelocyticleukemias, to the effect on differentiation of retinoic acid or othercompounds normally capable of inducing cell maturation but for whichphenomena of resistance by the cells concerned may be observed, the saidprocedure being characterized in that it comprises the placing of aspecific quantity of the molecule under study in contact with aresistant cell line such as defined above, followed by the detection ofpossible differentiation of the cells of the said line in the presenceof retinoic acid or of the other compounds mentioned above.

The above-mentioned procedure advantageously makes it possible to selectmolecules which are capable of abolishing this phenomenon of resistancementioned above, on the one hand, and of restoring cellular maturationunder the above-mentioned conditions, on the other.

In the context of the procedures mentioned above, the detection of thedifferentiation of the promyelocytes of the cell lines of the inventiontowards the granulocytic stage is advantageously carried out byapplication of one or more of the following methods.

Since this differentiation is expressed in particular by the activationof the expression of certain specific genes, the expression products ofthese genes are detected with the aid of specific antibodies, theseantibodies being labelled if necessary, in particular radioactively orenzymatically.

When these expression products exhibit an enzymatic activity or whenthis maturation is expressed by a disappearance of an enzymatic activitysuch as myeloperoxidase activity, the detection of the differentiationis advantageously achieved with the aid of substrates, labelled ifnecessary, in particular radioactively or enzymatically, on which theenzymatically active products expressed, or which are no longerexpressed, are capable of acting.

The detection of this cellular differentiation may also be carried outby a morphological study of the cells, in particular after staining ofthe latter. As an example of a staining method for the cells, mentionmay be made of the May-Grunwald-Giemsa stain.

Cellular differentiation may also be assessed according to a functionalcriterion, i.e. a study is made as to whether the differentiated cellsare functional in the same way as normal granulocytes. As an example ofa test capable of making possible the detection of functional criteriaof normal granulocytes, mention should be made of the NBT (Nitro BlueTetrazolium) test (Pick E., Methods Enzymol. 133:407, 1986).

The response time of the cell lines of the invention to the moleculesunder study when used in the procedures described above varies fromseveral hours to several days depending on the method of detection ofcellular differentiation selected.

As an illustration, the incubation time necessary before detection ofthe activation of the expression of genes is of the order of 30 minutesto 24 hours, that necessary prior to detection of the modification ofthe morphological aspect of the cells varies from 15 hours to 5 days,and that necessary prior to detection of the functional aspect of thecells varies from 3 days to 7 days.

Advantageously, these screening methods make it possible to selectmolecules capable of being used in the area of the prevention ortreatment of diseases caused by an abnormal proliferation of the cells,in particular malignant tumors in general, and more particularlyleukemias, especially acute promyelocytic leukemias.

More particularly in the case when cell lines resistant to retinoic acidare used, these methods make possible the selection of molecules capableof being used in the context of the prevention of the appearance ofresistance phenomena directed against the differentiation effect ofretinoic acid or other compounds normally capable of inducing cellmaturation of the cells of a patient when this latter is being treatedwith retinoic acid or compounds mentioned above.

Still in the context of the use of cell lines resistant to retinoicacid, these procedures according to the invention make possible theselection of molecules capable of being used in the context of thetreatment of patients in relapse after treatment with retinoic acid orother compounds normally capable of inducing cell maturation.

The invention also relates to the cultures themselves constituted bycells of the cell lines of the invention, particularly the NB4 or NB4Rcells mentioned above, and a nutrient support enabling the cells toproliferate, in particular the RPMI 1640 medium supplemented with fetalcalf serum indicated above.

Consequently, the subject of the invention is kits for theimplementation of the screening procedures such as those describedabove, these kits containing:

a culture such as that described above comprising a cell line accordingto the invention,

suitable reagents for the detection of the possible differentiation ofthe cells, in particular:

* antibodies capable of recognizing specifically the polypeptidesproduced by the activation of the expression of certain genes, theseantibodies being labelled if necessary, in particular radioactively orenzymatically,

* substrates, labelled if necessary, in particular radioactively orenzymatically, on which the enzymatically active products, expressed orwhich are no longer expressed, are capable of acting,

* enzymes, labelled if necessary, in particular radioactively orenzymatically, and capable of acting on substrates produced duringcellular differentiation,

* cell stains.

The cell lines of the invention are also characterized in that thesurface membranes of the cells constituting them are recognized inparticular by all of the antibodes directed specifically againstantigens of human leukocyte membranes of class I (HLA class I), themarkers CD13 and CD33 of the myeloid cells, the markers CD15 and CD11bof the granulocytes, the markers CD9 and CD11B of the monocytes, themarker CD11b of alpha-integrin, the marker CD38 of the activated Tcells, the marker CD2 of the T cells, the marker CD4 of the HIV receptorof the T helper cells, respectively, or are recognized by some of themonly, but are not recognized by the antibodies directed against theantigens of human leukocyte membranes of class II (HLA class II), themarker CD10 of the antigens of the leukocyte surface membrane of acutelymphocytic leukemias (CALLA), the markers CD11c, CD14, CD36 of themonocytes, the marker CD36 of the platelets, the marker CD7 of theimmature T cells, the marker CD3 of the T cells, the beta chain of theIL-2 cell receptor, the marker CD19 of the B Pan cells, the CD23 markerof the mature B cells, the marker CD34 of the precursor cells of thelymphocytes and myelocytes, the marker CD41 of the platelets of groupII/IIIa, the marker CD42 of the platelets of group IX, the erythrocytes,glycophorin, or are not recognized by some of them only.

The presence of the marker CD4 at the surface of the cells of theinvention confers on these latter the property of being a model ofchoice for the study of the process of the infection of the cells by theviruses of the HIV type.

The subject of the present invention is also the use of theabove-mentioned cell lines as positive controls in the context of invitro diagnostic procedures for acute human promyelocytic leukemias oralso the use of the resistant cell lines described above as positivecontrols in the context of in vitro diagnostic procedures for thepossible appearance of cells resistant to retinoic acid or othercompounds capable of inducing the cell maturation during the treatmentof a patient with these compounds.

The invention also relates to kits for the implementation of the invitro diagnostic procedures for acute human promyelocytic leukemias,these kits containing a cell line selected from those described above aspositive control of the presence of cells characteristic of acute humanpromyelocytic leukemias.

The invention also relates to kits for the implementation of in vitrodiagnostic procedures for the possible appearance of cells resistant toretinoic acid or other compounds capable of inducing cell maturationduring the treatment of a patient with these compounds, these kitscontaining a resistant cell line selected from those described above aspositive control of the presence of cells resistant to the compoundsmentioned above.

The subject of the present invention is also polypeptides produced bythe cell lines of the invention, whether they are secreted or not, andimplicated in the mechanism of autonomous proliferation of the cells ofthese lines.

These polypeptides, growth factors for example, are obtained from thecell lines of the invention, in particular according to the methodcomprising the following steps:

incubation of the cells of the lines of the invention in a culturemedium,

withdrawal of a sample of the incubation medium and detection of thepossible presence of one or more polypeptides such as defined secretedinto the medium with the aid of a proliferation and differentiation testcarried out on reference hematopoietic cells, or on the lines NB4 andNB4R or their variants,

biochemical separation (chromatography, electrophoresis, etc. . . ) ofthe polypeptide(s) mentioned above.

These steps described above lead to the production of the naturalmolecule(s).

The production of the recombinant molecule(s) may be carried out byscreening an expression library of the lines NB4 and NB4R or a variantof these lines producing the desired biological activity, followed bygenetic cloning and the production of the above-mentioned recombinantmolecule(s).

The subject of the invention is more particularly the growth factor(s)derived from the cell lines NB4 and NB4R, and capable of being obtainedaccording to the procedure indicated above.

The invention will be illustrated more particularly with the aid of thedetailed description which follows of the production of the cell lineNB4 and the cell line NB4R.

I MATERIALS AND METHODS

a) Cell culture and establishment of the cell line NB4

A sample of bone marrow was taken from a patient suffering from APLafter this patient had received treatment with retinoic add. Theleukocytes were separated by centrifugation on Ficoll-Hypaque andcultured at a concentration of 10⁴ cells/ml on a nutrient supportconstituted of cells of human bone marrow stroma (Lanotte et al., J.Cell Sci. 50:281, 1981), with a RPMI 1640 medium (GIBCO, Grand Island,N.Y.) supplemented with 12.5% fetal calf serum (FCS), 7.5% horse serum(Flow Lab, Scotland) in a plastic bottle at 37° C. in moist air plus 5%CO₂. The cultures were maintained by replacing one half of the growthmedium each week with fresh medium. After 4 to 5 weeks, the adherentlayer containing regions of proliferating leukemic cells was detachedmechanically and the entire cell population was transferred withoutdissociation to a bottle containing a fresh nutrient support consistingof cells of bone marrow stroma. In this way a progressive enrichment ofproliferating APL cells was obtained. Development was observed duringfourteen weeks, depending on the microenvironment. Autonomous growth wasthen detected and a rapidly developing cell population invaded theculture and became a cell line. The APL cells thus obtained aredesignated NB4. The NB4 growth conditions were improved by culturing thecells at a concentration of 2×10⁵ cells/ml in a RPMI 1640 mediumsupplemented with 10% FCS only (the doubling time varies from 36 to 40hours). Aliquot quantities of cells were frozen at -80° C.

b) Cytogenetic analysis

The chromosomes were studied when the cell line had been established andafter 40 weeks of culture with 2 passages per week. The R bandsprocedure (RHG) was used and the chromosomes were classed according tothe international nomenclature (ISCN/1985/: An International System forHuman Cytogenetic Nomenclature) (Harnden D. G., Klinger H.P./eds/Published in collaboration with Cytogenet Cell Genet. Basel,Switzerland, Karger, 1985).

c) Induction of differentiation

The NB4 cells (10⁵ cells/ml) in RPMI medium containing 10% FCS andvariable concentrations of all-trans retinoic acid (Sigma) as inducingagent were incubated for 1 to 5 days on culture microplates. Thematuration was assessed by microscopic examination of the enzymaticstaining on the cytological plates and also by the adhesion of the cellsof the stroma to the extracellular matrix, fibronectin. The cells werestained with the aid of May-Grunwald-Giemsa. The reactions were carriedout with myeloperoxidase, alpha-naphthyl butyrate esterase, naphthol-ASDchloroacetate esterase, alkaline phosphatase and with NBT.

d) Cell surface markers

The staining of the cell suspensions in the case of indirectimmunofluorescence was carried out according to the methods described in(Chen Z. et al.: Immunological typing of ALL: Concurrent analysis byflow cytometry and immunocytology. Leuk Res. 10:1411, 1986). Thecytofluorometric analysis was carried out on an EPICS profile(Coultronics, Margency, France). A large number of monoclonal antibodieswas used (cf. above).

II RESULTS

a) Establishment, morphology and cytometry of the cell line.

The APL cells were cultured on a layer of cells of the stroma of bonemarrow. At the start of the culture a sub-population of cells havingachieved a certain "cooperation" with the hematopoietic microenvironmentwas selected. The total population, with interaction losses with thecells of the stroma, was constituted of hypergranular promyelocytes inthe G0/G1 phase, whereas some leukemic cells strongly associated withthe cells of the stroma had a mitotic activity and were much lessgranular with the morphology of the blast cells found in the APLs. Atthe time when the NB4 cell line was isolated, only the blast cellsproliferated. After 9 months of culture (i.e. about 155 cell doublings)no significant change was observed.

b) Cytogenetic study

The cytogenetic analyses were performed at the initial stage of theculture when the NB4 cell line was isolated, and after nine months ofculture. At the initial stage of culture, the 23 metaphases examinedwere abnormal with caryotypic variations from one cell to another. Thenumber of their chromosomes varied between 68 and 90, most of them beinglocated in the hypotetraploid region, with random losses.

All of the metaphases have the translocation t (15; 17) (q22; q11-12)associated with other rearrangements varying from one cell to another.However, a loss of chromosome 19 and a replacement by 19q+, on the onehand, and a der (12; ?) (p 12; ?) was present in all of the metaphasesexamined.

The translocation t (15; 17) present in the first caryotype conducted onthe patient suffering from APL mentioned above and at the stage of hissecond relapse was observed in all of the metaphases of the establishedcell line. The caryotypic complexity which was observed in the cultureof bone marrow in the short-term was found in the cell line with a greatvariation from one cell to another. The caryotype was hypotetraploidwith loss of at least one copy of the chromosomes 8, 11 and 14. Arepresentative caryotype may be summarised in the following manner:

80-87, XXX, -X, -3, -8, -9, -10, -12, -14, -14, -18, -19, -19, der (12),t (12;?) (p12;), t (15; 17) (q22; q11-12), t (15; 17), der (19) t (19;?), + variable markers.

In view of this variation, it is difficult to establish the minordifferences between the caryotypes of the "fresh" cells and thecaryotypes of the cells of the NB4 line. However, it was possible todetect a new marker der (12) in the cell line whereas it was not presentin the "fresh" cells. The rearrangement of the chromosome 19, resemblingHSR (19) (q13), which is found in certain metaphases of the "fresh"cells, was apparently selected during the establishment of the cell linesince it was present in all of the metaphases, most of the time induplicate.

The caryotype of the NB4 line is shown in FIG. 2.

c) Immunocytological analysis

The cell surface markers of NB4 were analysed (cf.above) and monitoredfor 9 months, but no significant change was detected. It was thusdetermined that NB4 expresses specific markers at the granulocyticstage, but also lymphoid cell markers such as CD2, CD4 and a monocytemarker CD9. The percentages of the positive cells compared with themyeloid markers (73% to 89%) and T cell markers (73% for CD4) suggestthat the characteristics of the two lines are expressed simultaneously.CD9 is expressed by 73% of the cells, whereas the cells are clearlynegative with respect to the other markers associated with the monocytesuch as CD14 and CD36. The expression of these antigens might beassociated with activations of genes linked to the alterations ofmultiple caryotypes which were found.

d) Retinoic acid induces maturation

NB4 cells were treated with trans retinoic acid (1 umol/l) for six days.The cessation of cellular proliferation occurred 48 hours later;morphological maturations accompanied by modifications of the surfaceantigens and functional markers can be detected. A large increase in themarker correlated with alpha-integrin, CD11b (Arnaout M. A.: Structureand function of the leukocyte adhesion molecules CD11 CD18. Blood75:1037, 1990) was observed; CD11c, which is absent from blast cells isstrongly expressed on 75% of the differentiated cells; these changes areassociated with a marked increase in the adhesion of the cells to theextracellular matrices. The production of superoxide and hydrogenperoxide, and the reduction of the NBT were analysed quantitatively byELISA and by cytochemical methods; a large increase in the positivity ofthe NBT test indicates a potential and microbial capacity confirming themorphological maturation induced by the retinoic acid.

III ANALYSIS OF THE RESULTS

The NB4 cell line is the only permanent line exhibiting a translocationt (15; 17) which has been established from leukemic cells of a patientsuffering from APL.

The cells derived from human APLs (M3) exhibit a remarkable manner ofproliferating in vitro which is the probable reason for the failureshitherto to obtain a permanent cell line. It may be concluded from thesefailures: (1) that the APL cells have a very low proliferation potentialin vitro. The analysis of the cell cycle by means of cytometry shows acessation of the proliferation in the G1 phase (or possibly in phase G0)for several weeks, during which the cells become hypergranular andexhibit an intense myeloperoxidase reaction. However, the APL cellssurvive in culture, whereas many cellular types of leukemias die; thatsuggests that these cells do not require survival factors, as distinctfrom normal promyelocytes or cell lines dependent on factors. (2) noneof the hematopoietic growth factors tested so far (including G-CSF)leads to in vitro proliferation for more than 3 to 4 cycles. (3) the APLcells undergo cell-cell or cell-matrix interactions with the cells ofthe stroma of the bone marrow. Some APL cells associated with themicro-environment (1 to 10 per 10⁵ cells) proliferate.

In the light of what has just be stated it might be expected that APLcells co-cultured with bone marrow stroma for several months could leadto the selection of cells capable of proliferating. The authors of thepresent patent application have put forward the hypothesis that theisolation of a permanent APL cell line depends on a second eventresponsible for the autonomy of proliferation and that its probabilityshould be considerably increased in a population undergoing cyclicgrowth as compared to a population arrested at stage G1. This strategywas adopted in order to isolate the NB4 cell line. The primary cultureof leukemic cells on a layer of stroma cells provides a favourableenvironment for cell proliferation. It cannot be asserted that anadditional mutation is responsible for the appearance of autonomousleukemic cells which develop in the culture of APL cells depending onthe microenvironment. It should be emphasized that an additional markerder (12) is present in the cell line but cannot be detected in the"fresh" cells. On the other hand, a self-renewing cell population mightalready be present in the bone marrow of the patient and would becapable of being selected in vitro. Several observations support thislatter hypothesis: (1) Among the multiple chromosomal changes in theblast cells of the patient, which are perhaps partially related to priortreatments, some rearrangements favouring in vitro proliferation mayhave been selected. (2) The NB4 cells have conserved morphologicalcharacteristics of a minority population of blast cells which was alsofound in the patient at the time of his relapse.

The retinoic acid induces a rapid maturation of the NB4 cells from themorphological and functional point of view; after 3 days of continuoustreatment with retinoic acid cell proliferation is no longer detectable.The maturation of the NB4 cells with other inducers such as phorbolesters, dimethyl sulfoxide, cyclic nucleotides and corticosteroids neverequals in intensity and rapidity the maturation induced by retinoicacid.

FIG. 1 shows the DNA fingerprint of the NB4 cell line, and which isobtained by digestion of the genome of the cells with HinfI, followed bymigration of the DNA fragments obtained in gel electrophoresis anddetection of fragments with the aid of the probe M13 (cf. Nucleic AcidsResearch mentioned above).

FIG. 2 shows the caryotype of the NB4 cell line, the arrows indicate therearranged chromosomes.

IV ESTABLISHMENT OF THE NB4R LINE

Cells of the initial isolate of the human promyelocytic line NB4 wereincubated in the continuous presence of all-trans retinoic acid: afterseveral months of culture, some resistant cells were isolated andamplified constantly in the presence of retinoic acid. This isolate hasmade it possible to isolate a NB4 cell line resistant to retinoic acid(NB4R line). The resistant cells constitute an autonomous line,permanent in the same way as the sensitive NB4 line and which can bemaintained under the same conditions. It is interesting to note thatthis resistance corresponds to a commonly observed clinical fact, namelythe relapse after a quite prolonged remission following treatment withretinoic acid. The patient from whom these lines are derived was himselfin relapse after chemotherapy and treatment by retinoic acid. Hence itis possible to say that the two isolates NB4 and NB4R, one sensitive toretinoic acid, the other resistant, represent two parts of this disease.These cells make it possible to study the mechanism of acquisition ofresistance to the treatment of promyelocytic leukemias by retinoic acid.This complementarity reinforces the interest in the tool which theselines constitute for the study of the effects of the therapeutic agentsand for the research of agents to prevent (or abolish) resistance tothese agents. The resistant line constitutes the only existingbiological model of a promyelocytic line with t (15; 17) enablingmolecular screening to be carried out in order to discover novelmedicines active on the cells of patients in relapse after treatmentwith retinoic acid.

We claim:
 1. A permanent cell line of human promyelocytic cellsdesignated NB4, wherein the cell line NB4 was deposited with theCollection Nationale de Culture de Micro-organismes de l'INSTITUTPASTEUR (CNCM) on Feb. 25, 1991 under accession number I-1045, and withthe Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSM)on Feb. 14, 1992 under accession number DSM.ACC.2030.
 2. The cell lineaccording to claim 1, wherein the cells exhibit DNA fingerprints on anelectrophoresis gel identical, in whole or in part, with the DNAfingerprint shown in FIG.
 1. 3. The cell line according to claim 1,wherein the cell line comprises a cell culture in association withnutrient elements enabling cells of the cell culture to proliferate. 4.A kit for a method for in vitro screening of acute human promyelocyticleukemia utilizing an NB4 cell line as a positive control, wherein thecell line NB4 was deposited with the Collection Nationale de Culture deMicro-organismes de l'INSTITUT PASTEUR (CNCM) on Feb. 25, 1991 underaccession number I-1045, and with the Deutsche Sammlung vonMikro-organismen und Zellkulturen GmbH (DSM) on Feb. 14, 1992 underaccession number DSM.ACC.2030, said kit comprising:(a) an NB4 cell line;and (b) a reagent suitable for detection of cellular differentiation.